JP4550202B2 - Sugar chain containing 1,5-D-anhydrofructose as a constituent sugar - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel sugar chain containing 1,5-D-anhydrofructose as a constituent sugar and a method for producing the same.
[0002]
[Prior art]
A sugar chain is a compound having a structure in which two or more monosaccharides are dehydrated and condensed. Up to now, various sugar polymers including starch can be decomposed by chemical and enzymatic treatment, monosaccharides can be polymerized by chemical and enzymatic treatment, or a single sugar chain can be used. They are synthesized using a method of transferring sugar to one sugar chain, and some have been industrialized. Many of these sugar chains are composed of glucose, xylose, or epimers or derivatives thereof, and there are no reports on sugar chains containing an anhydro sugar such as 1,5-D-anhydrofructose as a constituent sugar.
[0003]
1,5-D-anhydrofructose is produced using starch or starch degradation product as a substrate by the action of α-1,4 glucan lyase, an enzyme present in plant tissues such as basidiomycetes or red algae. Can do. 1,5-D-anhydrofructose has an interesting and unique structure in which glucose is dehydrated. From the results of the analysis so far, it has become clear that it has characteristic properties such as high reducing power and antibacterial activity compared to other monosaccharides such as glucose.
[0004]
[Problems to be solved by the invention]
An object of the present invention is to provide a novel sugar chain containing 1,5-D-anhydrofructose as a constituent sugar.
[0005]
Another object of the present invention is to provide a method for producing a novel sugar chain containing 1,5-D-anhydrofructose as a constituent sugar.
[0006]
Still other objects and advantages of the present invention will become apparent from the following description.
[0007]
[Means for Solving the Problems]
According to the present invention, the above objects and advantages of the present invention include, firstly , 1,5-D-anhydrofructose as a constituent sugar at the reducing end.
A sugar chain having the formula
G- (G) _n -AF
Where G is a glucose skeleton, AF is a 1,5-D-anhydrofructose skeleton, and n is a number from 0 to 20, provided that the glucose skeleton has other glucose units as side chains. May be glycosidically bonded,
It is achieved by a method for producing a sugar chain characterized by:
[0008]
Further, the above objects and advantages of the present invention, the second, the cyclodextrin and 1, 5-D-anhydrofructose to brought the contact in the presence of cyclodextrin synthetase as transglycosylation catalytic cyclodextrin A sugar chain containing 1,5-D-anhydrofructose as a constituent sugar at the reducing end, wherein the monosaccharide unit or the partial sugar chain unit constituting the sugar is transferred to 1,5-D-anhydrofructose Achieved by the method of manufacturing.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the production method of the present invention will be described first.
[0010]
The raw material 1,5-D-anhydrofructose is obtained by decomposing starch or glycogen with α-1,4-glucan lyase.
[0011]
It has been reported that α-1,4-glucan lyase is contained in seaweeds and mushrooms. The present inventors purified α-1,4-glucan lyase from seaweed ogonori and synthesized 1,5-D-anhydrofructose by allowing this enzyme to act on starch (for example, waxy corn starch). This 1,5-D-anhydrofructose was purified and used for sugar chain synthesis.
[0012]
In addition, as the other raw material polysaccharide, for example, glucans are preferably used. Of these, sugars having an α-1,4-glucan chain such as cyclodextrin, starch, soluble starch, maltodextrin, and derivatives thereof are more preferable.
[0013]
These raw materials are contacted in the presence of a sugar chain transfer catalyst. The transglycosylation catalytic, cyclo dextrin synthase is used.
[0014]
For example, the enzymatic reaction of sugar chain synthesis can be carried out by causing cyclodextrin synthase to act on 1,5-D-anhydrofructose and α-1,4-glucan such as starch. In addition to cyclodextrin synthesis, cyclodextrin synthase catalyzes a sugar chain transfer reaction. Therefore, when this enzyme is allowed to act in the presence of 1,5-D-anhydrofructose and α-1,4-glucan, sugars having various degrees of polymerization are added to the non-reducing end of 1,5-D-anhydrofructose. It is possible to synthesize a sugar chain having 1,5-D-anhydrofructose as a constituent sugar.
[0015]
The above sugar chain synthesis reaction is preferably carried out in an aqueous medium. As the aqueous medium, water or, optionally, a mixed medium composed of water and a water-miscible organic medium such as alcohol is used. The reaction temperature and pH vary depending on the type of glycan transfer catalyst used, but can be carried out in the range of 10 to 65 ° C. and pH of 3 to 8, for example.
[0016]
Moreover, the density | concentration of the substrate which is a raw material can be 1-40 g / 100 ml, for example. After completion of the reaction, usually after inactivating the enzyme used, or when the reaction is carried out by immobilizing the enzyme on a carrier, the reaction solution is contacted with the enzyme, and then the product is obtained according to a conventional method. Can be separated. Thus, according to the present invention, a sugar chain (sugar molecule chain) containing 1,5-D-anhydrofructose as a constituent sugar is produced and provided.
[0017]
The sugar chain of the present invention containing 1,5-D-anhydrofructose as a constituent sugar is other than 1,5-D-anhydrofructose having 1,5-D- anhydrofructose at the reducing end of the sugar chain . part is the is not preferred maltodextrin sugar chain.
[0018]
According to the invention, in particular the following formula G- (G) _n -AF
Where G is a glucose skeleton, AF is a 1,5-D-anhydrofructose skeleton, and n is a number from 0 to 20, provided that the glucose skeleton has other glucose units as side chains. May be glycosidically bonded.
The sugar chain represented by is preferably provided.
[0019]
The novel sugar chain of the present invention can be used as a sugar chain donor that reacts with various compounds having an amino group, for example, an amino group of a protein to bind the sugar chain to the amino group.
[0020]
Hereinafter, the present invention will be described in more detail by way of examples.
[0021]
【Example】
Example 1
After mixing 100 mg of 20 mg / ml 1,5-D-anhydrofructose and 100 ml of 50 mg / ml β-cyclodextrin to adjust the pH to 5, add 50 U of cyclodextrin synthase to the mixed solution at 35 ° C. The reaction was allowed to proceed for 48 hours. After completion of the reaction, the reaction solution was heated to 95 ° C. to deactivate the enzyme. As a result of analyzing the reaction solution by HPLC (TOSO NH2-60), it was revealed that sugar chains having a polymerization degree of 2 to 11 were synthesized as shown in FIG.
[0022]
Next, an experiment was performed to confirm that these sugar chains contain 1,5-D-anhydrofructose. First, the sugar chain of each polymerization degree was fractionated from said reaction product by gel filtration chromatography. Hydrochloric acid was added to the separated sugar chains of each degree of polymerization so that the final concentration was 0.5N, and the mixture was heated in a boiling bath for 30 minutes to hydrolyze hydrochloric acid, and the constituent sugars were analyzed by high performance liquid chromatography (HPLC). In the sugar chain having a polymerization degree of 2, two peaks having a quantitative ratio of 1: 1 were detected at the same retention time as the standard sample of glucose and 1,5-D-anhydrofructose (FIG. 2). In the sugar chain having a polymerization degree of 3, two peaks having a 2: 1 ratio of glucose to 1,5-D-anhydrofructose were detected at the same retention time as that of each standard sample (FIG. 3). Furthermore, as a result of examining sugar chains having a polymerization degree of 4, 5, 6 or more (polymerization degree n) in the same manner, similarly, glucose versus 1,5-D-anhydrofructose at the same retention time position as each standard sample. Two peaks with an amount ratio of (n-1): 1 were detected. The above results indicate that the reaction product contains one 1,5-D-anhydrofructose residue per molecule. Next, glucoamylase was allowed to act on a mixture of sugar chains of each degree of polymerization, which is a cyclodextrin synthase reaction product.
As a result, as shown in FIG. 4, glucose and a sugar having a polymerization degree of 2 were obtained. Glucoamylase is an enzyme characterized by degrading one glucose residue at a time from the non-reducing end of a glycosidic-linked glucose chain. Therefore, these sugar chains indicate that the portion other than the sugar having a polymerization degree of 2 is composed of glucose, and that the non-reducing end of the sugar having a polymerization degree of 2 is glucose. Next, the hydrolysis product of the sugar having a polymerization degree of 2 was analyzed by HPLC. As a result, two peaks in which the ratio of the former to the latter was 1: 1 were detected at the same retention time positions as the standard samples of glucose and 1,5-D-anhydrofructose. Furthermore, the solution at each peak position was fractionated and analyzed. When the substance detected at the same position as the retention time of glucose was quantified by a glucose oxidase method generally used for quantifying glucose, the same amount of glucose as estimated from the peak area was detected. Since glucose oxidase is an enzyme highly specific for glucose, this result indicates that the substance detected at the same position as the retention time of glucose is glucose.
On the other hand, when the substance detected at the same position as the retention time of 1,5-D-anhydrofructose was analyzed by NMR, 1,5-D-anhydro having a purity of 99% shown in FIG. Fructose standard sample pattern (1,5-D-anhydrofructose dissolved in heavy water to 10% and measured with a JEOL GSX-500 spectrometer. Chemical shifts are expressed in ppm using tetramethylsilane as a reference substance. The same pattern as that of 1,4-dioxane (67.40 ppm) was used as a control as an external standard substance. Therefore, it is shown that the substance detected at the same position as the retention time of 1,5-D-anhydrofructose is 1,5-D-anhydrofructose. From the above results, it can be concluded that the synthesized sugar chain is composed of 1,5-D-anhydrofructose at the reducing end, and glucose is glycosidically bound thereto.
[0023]
As a raw material for synthesizing a sugar chain containing 1,5-D-anhydrofructose, α-1, cyclo-dextrin and other cyclic oligosaccharides such as α-cyclodextrin and γ-cyclodextrin, starch, soluble starch and the like α-1, Sugars containing 4-glucan or derivatives thereof can be used. For example, when 1,5-D-anhydrofructose and soluble starch are mixed at a ratio of 1 to 2, 10 U of cyclodextrin synthase is added per 1 g of soluble starch and reacted at a temperature of 35 ° C. for 48 hours, 1,5-D- About 50% of anhydrofructose was converted to a sugar chain.
[0024]
Example 2
When 1,5-D-anhydrofructose and soluble starch are mixed at a ratio of 1 to 2, 10 U of cyclodextrin synthase is added per 1 g of soluble starch and reacted at a temperature of 35 ° C. for 48 hours, 1,5-D-anhydro is obtained. About 50% of the fructose was converted to sugar chains. As a result of examining the constituent sugar of this oligosaccharide in the same manner as in Example 1, it was a sugar chain having a degree of polymerization of 2 to 10 containing 1,5-D-anhydrofructose at the reducing end.
[Brief description of the drawings]
FIG. 1 is an HPLC chart (TOSO NH2-60) after completion of an enzymatic reaction with cyclodextrin synthase.
FIG. 2 is an HPLC chart (Mitsubishi Kasei CK08S) of a hydrolysis product obtained by hydrolyzing an oligosaccharide having a polymerization degree of 2 with hydrochloric acid.
FIG. 3 is an HPLC chart (Mitsubishi Kasei CK08S) of a hydrolysis product obtained by hydrolyzing an oligosaccharide having a polymerization degree of 3 with hydrochloric acid.
FIG. 4 is an HPLC chart (Mitsubishi Kasei CK08S) of a reaction solution obtained by treating a sugar chain having a polymerization degree of 3 with glucoamylase.
FIG. 5 is an NMR chart of 1,5-D-anhydrofructose having a purity of 99% or more.

Claims (4)

Contains 1,5-D-anhydrofructose as a constituent sugar at the reducing end
A sugar chain having the formula
G- (G) _n -AF
Where G is a glucose skeleton, AF is a 1,5-D-anhydrofructose skeleton, and n is a number from 0 to 20, provided that the glucose skeleton has other glucose units as side chains. May be glycosidically bonded,
A sugar chain represented by the formula: The sugar chain according to claim 1, wherein the constituent sugar moiety other than 1,5-D-anhydrofructose is a maltodextrin sugar chain. A monosaccharide unit or part constituting a polysaccharide by contacting an oligosaccharide, a cyclic oligosaccharide or a polysaccharide with 1,5-D-anhydrofructose in the presence of cyclodextrin synthase as a sugar chain transfer catalyst A method for producing a sugar chain containing 1,5-D-anhydrofructose as a constituent sugar at the reducing end, wherein a sugar chain unit is transferred to 1,5-D-anhydrofructose. Cyclodextrin and 1,5-D-anhydrofructose are contacted in the presence of cyclodextrin synthase as a transglycosylation catalyst, and the monosaccharide unit or the partial sugar chain unit constituting the cyclodextrin is converted to 1,5. -A method for producing a sugar chain containing 1,5-D-anhydrofructose as a constituent sugar at the reducing end , which is transferred to D-anhydrofructose.

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